Anti-biofilm properties of a mupirocin spray formulation against Escherichia coli wound infections.

Mupirocin ointment is a widely used topical drug for the treatment of bacterial skin infections. However, ointments have some limitations which motivated the development of a film forming spray of mupirocin. Mupirocin spray (2%) was formulated with Eudragit E100 as a film forming agent and tested for its antibacterial and anti-biofilm activities against Escherichia coli, a skin pathogen causing wound and surgical site infections. Treatment with mupirocin spray resulted in significant antibacterial and anti-biofilm activities (inhibition and disruption) with single spray and sub-actual dose concentrations at par with the commercial ointment concentration. The spray formulation was found to be non-toxic to fibroblast cells and greatly resisted removal from the site of application upon washing, in contrast to the ointment which was significantly removed after a single wash. This is the first study to develop and evaluate a spray formulation for mupirocin that forms a stable thin film for sustained release of the drug.

Anti-biofilm mechanisms of 3,5-di-tert-butylphenol against clinically relevant fungal pathogens.

The methanolic extract (PFME) of Pleurotus florida was assessed for anti-biofilm activity against Candida species. 3,5-Di-tert-butylphenol (3,5-DTB) was identified as the major antifungal constituent in PFME. In its pure form 3,5-DTB inhibits, disrupts, and reduces the viability of biofilm cells as seen from scanning electron and confocal microscopy studies. Microscopic studies and propidium iodide uptake assays confirmed that 3,5-DTB damages the cell membrane of Candida cells. In addition, 3,5-DTB induces accumulation of reactive oxygen species (ROS) which contribute to its pronounced anti-biofilm activity. The results of the present study show that 3,5-DTB exhibits combined anti-biofilm and conventional fungicidal activity against Candida species and elucidate the underlying mechanisms.

Role of Indole Production on Virulence of Vibrio cholerae Using Galleria mellonella Larvae Model.

Cell to cell communication facilitated by chemical signals plays crucial roles in regulating various cellular functions in bacteria. Indole, one such signaling molecule has been demonstrated to control various bacterial phenotypes such as biofilm formation and virulence in diverse bacteria including Vibrio cholerae. The present study explores some key factors involved in indole production and the subsequent pathogenesis of V. cholerae. Indole production was higher at 37 °C than at 30 °C, although the growth at 37 °C was slightly higher. A positive correlation was observed between indole production and biofilm formation in V. cholerae. Maximum indole production was detected at pH 7. There was no significant difference in indole production between clinical and environmental V. cholerae isolates, although indole production in one environmental isolate was significantly different. Both growth and indole production showed relevant changes with differences in salinity. An indole negative mutant strain was constructed using transposon mutagenesis and the direct effect of indole on the virulence of V. cholerae was evaluated using Galleria mellonella larvae model. Comparison to the wild type strain, the mutant significantly reduced the mortality of G. mellonella larvae which regained its virulence after complementation with exogenous indole. A gene involved in indole production and the virulence of V. cholerae was identified.

Effect of 2, 4-di-tert-butylphenol on growth and biofilm formation by an opportunistic fungus Candida albicans.

Candida albicans, an opportunistic pathogen, has been known to form hypoxic biofilms on medical devices which in turn confers resistance towards antifungals, resulting in subsequent therapeutic failures. Inclusion of anti-biofilm agents in the control of infections is a topic of current interest in developing potential anti-infectives. The in vitro anti-fungal and anti-biofilm efficacy of 2,4-di-tert-butyl phenol [DTBP] was evaluated in this study, which revealed the potential fungicidal action of DTBP at higher concentrations where fluconazole failed to act completely. DTBP also inhibited the production of hemolysins, phospholipases and secreted aspartyl proteinase which are the crucial virulence factors required for the invasion of C. albicans. Various anti-biofilm assays and morphological observations revealed the efficacy of DTBP in both inhibiting and disrupting biofilms of C. albicans. Inhibition of hyphal development, a key process that aids in initial adhesion of C. albicans, was observed, and this could be a mechanism for the anti-biofilm activity of DTBP.

Burdock root extracts limit quorum-sensing-controlled phenotypes and biofilm architecture in major urinary tract pathogens.

Bacterial biofilms are serious concern in patients infected with urinary tract infections, complicated urinary tract infections and other device-associated infections. Microbes within the biofilms are effectively shielded from antibiotics and host immune cells, hence can be treated only with agents which has the potential to disassemble the biofilms. The study is focused on the root extracts of Arctium lappa Linn. as a source for complementary medicine against three major biofilm forming clinical isolates of Escherichia coli, Proteus mirabilis, and Serratia marcescens. Methanol extracts of burdock roots (BR) showed no bactericidal activity (p > 0.05) against the uropathogens, whereas restrained the biofilms (p < 0.05) on polystyrene and glass surfaces at a biofilm inhibitory concentration of 100 µg/mL. The 3D confocal laser scanning microscopy was used to analyze the biofilm architecture which showed significant reduction in the surface area. Z-stack analysis has also revealed substantial reduction in the biofilm thickness (E. coli-50.79%, P. mirabilis-69.49%, and S. marcescens-75.84%). Further, BR extracts also inhibited quorum-sensing (QS)-controlled cellular phenotypes such as violacein, prodigiosin, swarming motility, and cell surface hydrophobicity. LC-MS/MS analysis of BR extracts identified the presence of two major quercetin derivatives (miquelianin and peltatoside) along with few other constituent components. Exploring such phytocompounds will provide potential agents to treat infections caused by biofilm forming uropathogens. The antibiofilm and anti-QS agents will ultimately serve as armor, facilitating the host immune system to fight infections.

Synergy of flavonoids with HDAC inhibitor: new approach to target Candida tropicalis biofilms.

Candida biofilms on various implanted medical devices and living tissues are serious concern in several hospital-acquired infections. The study was conducted to employ a new therapeutic strategy to inhibit the formation of Candida tropicalis biofilms. No significant antifungal or antibiofilm activity was observed when the sodium butyrate (SB), quercetin, and kaempferol were used as a lone drug. Significant decline (P<0·05) in biofilm formation was noted when sub-lethal concentration of SB (30 mM) was used in combination with the flavonoids (450 µg/ml). Z-stack analysis using 3D-confocal laser scanning microscopy (CLSM) also showed substantial reduction in the biofilm thickness in treated glass slides. In conclusion, this is the first report to our knowledge on implementing a combination therapy using flavonoids and histone deacetylase (HDAC) inhibitor.

The anti-biofilm potential of pomegranate (Punica granatum L.) extract against human bacterial and fungal pathogens.

Infectious diseases caused by bacteria and fungi are the major cause of morbidity and mortality across the globe. Multi-drug resistance in these pathogens augments the complexity and severity of the diseases. Various studies have shown the role of biofilms in multi-drug resistance, where the pathogen resides inside a protective coat made of extracellular polymeric substances. Since biofilms directly influence the virulence and pathogenicity of a pathogen, it is optimal to employ a strategy that effectively inhibits the formation of biofilm. Pomegranate is a common food and is also used traditionally to treat various ailments. This study assessed the anti-biofilm activity of a methanolic extract of pomegranate against bacterial and fungal pathogens. Methanolic extract of pomegranate was shown to inhibit the formation of biofilms by Staphylococcus aureus, methicillin resistant S. aureus, Escherichia coli, and Candida albicans. Apart from inhibiting the formation of biofilm, pomegranate extract disrupted pre-formed biofilms and inhibited germ tube formation, a virulence trait, in C. albicans. Characterization of the methanolic extract of pomegranate revealed the presence of ellagic acid (2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione) as the major component. Ellagic acid is a bioactive tannin known for its antioxidant, anticancer, and anti-inflammatory properties. Further studies revealed the ability of ellagic acid to inhibit the growth of all species in suspension at higher concentrations (>75 µg ml(-1)) and biofilm formation at lower concentrations (<40 µg ml(-1)) which warrants further investigation of the potential of ellagic acid or peel powders of pomegranate for the treatment of human ailments.

Anti-pathogenic Potential of Coral Associated Bacteria Isolated from Gulf of Mannar Against Pseudomonas aeruginosa.

Infections of Pseudomonas aeruginosa are of great concern because of its increasing resistance towards conventional antibiotics. Quorum sensing system of P. aeruginosa acts as a global regulator of almost all the virulence factors and majorly its biofilm formation. In the present study, quenching of QS system of P. aeruginosa has been explained with bioactives from bacteria associated with the coral Acropora digitifera. Isolated bioactives inhibited the expression of various virulence traits of P. aeruginosa like biofilm formation, and the production of extracellular enzymes like protease and elastase. This study also emphasises the potential of coral associated bacteria in producing bioactive agents with anti-pathogenic properties.

Inhibition of quorum sensing regulated biofilm formation in Serratia marcescens causing nosocomial infections.

Serratia marcescens is an opportunistic pathogen causing severe urinary tract infections in hospitalized individuals. Infections of S. marcescens are of great concern because of its increasing resistance towards conventional antibiotics. Quorum sensing (QS)-a cell to cell communication-system of S. marcescens acts as a global regulator of almost all the virulence factors and majorly its biofilm formation. Since, the QS system of S. marcescens directly accords to its pathogenesis, targeting QS system will provide an improved strategy to combat drug resistant pathogens. In the present study, QS system of S. marcescens has been used as target and its inhibition has been studied upon exposure to bioactives from coral associated bacteria (CAB). This study also emphasises the potential of CAB in producing bioactive agents with anti-QS and antibiofilm properties. Two CAB isolates CAB 23 and 41 have shown to inhibit biofilm formation and the production of QS dependent virulence factors like prodigiosin, protease, lipase and swarming motility. The study, on the whole explicates the potential of QS system as a target to treat drug resistant bacterial infections.

Role of aromatic stack pairing at the catalytic site of gelonin protein.

Aromatic-aromatic interactions play an important role in the enzyme-substrate recognition mechanism and in stabilization of proteins. Gelonin--a ribosome inactivating protein (RIP) from the plant Gelonium multiflorum--belongs to type-I RIPs and shows N-glycosylation activity which has been used as a model to explain the role of aromatic-aromatic stack pairing in RIPs. RIPs have a different substrate binding site and catalytic site. Role of tyrosine residues at the binding site has already been known but the role of tyrosine residues at catalytic site is still unclear. In this study, the role of tyrosine-adenine-tyrosine aromatic stack pairing at the catalytic site was studied by in silico mutation studies using molecular dynamic simulations. Through this study we report that, despite the fact that aromatic stack pairing aids in recognition of adenine at binding site, both the tyrosine residues of stack pairing play a crucial role in the stabilization of adenine at catalytic site. In the absence of both the tyrosine residues, adenine was unstable at catalytic site that results in the inhibition of N-glycosylation activity of gelonin protein. Hence, this study highlights the importance of π-π stack pairing in the N-glycosidic activity of gelonin by determining its role in stabilizing adenine at catalytic site.

In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant Staphylococcus aureus biofilms.

Staphylococcus aureus is now amongst the most important pathogenic bacteria responsible for bloodstream nosocomial infections and for biofilm formation on indwelling medical devices. Its increasing resistance to common antibiotics, partly attributed to its ability to form biofilms, is a challenge for the development of new antimicrobial agents. Accordingly, the goal of this study was to evaluate the effect of a coral associated actinomycete (CAA)-3 on S. aureus biofilms both in vitro and in vivo. Methanolic extracts of CAA-3 showed a reduction in in vitro biofilm formation by S. aureus ATCC 11632, methicillin resistant S. aureus ATCC 33591 and clinical isolates of S. aureus at the biofilm inhibitory concentration (BIC) of 0.1 mg ml(-1). Furthermore, confocal laser scanning microscope (CLSM) studies provide evidence of CAA-3 inhibiting intestinal colonisation of S. aureus in the nematode Caenorhabditis elegans. To conclude, this study for the first time, reports CAA as a promising source of anti-biofilm compounds, for developing novel drugs against highly resistant staphylococcal biofilms.

Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2.

The nucleotide sequence of a smallest cryptic plasmid pRK10 of Serratia marcescens ACE2 was determined. When compared to the all other plasmids reported so far from S. marcescens in sizes of over 70kb, pRK10 is only 4241bp long with 53% G+C content and has five coding sequences representing a coding percentage of 65.41. This small plasmid consists of one Tdh gene, four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. Analysis of the five open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization containing sequences homologous to the bom region and mobCABD genes of ColE1 and Tdh from Acinetobacter baumannii str. AYE. Results also indicate that pRK10 does not encode any gene for antibiotic/heavy metal resistance. Copy number and incompatibility of the plasmid with plasmids of ColE1 origin of replication was determined and it is quite stable in its natural host as well as in Escherichia coli DH5alpha. This relatively small plasmid will be useful for construction of shuttle vectors to facilitate the genetic analysis.